Early disease detection and disease monitoring are critical factors in diagnosing the correct physical symptoms and establishing the appropriate therapies. Plasma is frequently used as a material for the diagnosis, measurement and verification of diseases status. Therefore, it is an important body fluid to search for novel biomarkers not only to be used as a diagnostic tool, but also to elucidate new molecular pathways involved in diseases and mechanisms explaining altered homeostatic conditions.
Presently, plasma is only collected by invasive techniques. Whereas, plasma is the most important available source for systemic biomarkers obtained by invasive sources, other biological matrices have been explored to screen for biomarkers that are expected to operate at a more local level. Some examples of such alternative biological matrices are tumour tissue, cerebrospinal fluid and suction blister fluid.
The invasive extraction of suction blister fluid is largely derived from the interstitial fluid, which is the place where many important biomarkers are expected to be found. Interstitial fluid (or tissue fluid) is a solution which bathes and surrounds the cells of multicellular animals. It is the main component of the extracellular fluid, which also includes plasma and transcellular fluid. Compared to the very invasive skin biopsies used to analyze local mediators, suction blister fluid is obtained by means which allow less invasive extraction. Invasive suction blister fluid can be used as body fluid to detect small molecules such as glucose and lactate, drugs, study mediators of inflammation, etc. Because suction blister fluid is entrapped inside blisters, it cannot be used in conjunction with a sensor or a plurality of sensors for the continuous detection and/or the continuous monitoring for target molecule(s) since it cannot be in direct contact with any integrated sensor.
On the other hand, skin covers the entire external surface of the human body. Due to its size and accessibility, skin is an attractive target for a variety of applications. Most notably, transdermal route for delivering drugs has potential advantages over other methods of delivery in terms of convenience, non-invasiveness, and reduction of drug degradation. The predominant barrier to transdermal drug delivery is the outermost layer of the skin, the stratum corneum.
The stratum corneum is composed of several densely packed layers of flattened, dead, keratinized cells surrounded by lipid bilayers consisting primarily of ceramides, cholesterol, and free fatty acids. The total thickness of the stratum corneum varies from 10 to 40 μm with an average thickness of 20 μm. This strongly hydrophobic environment inhibits molecular transit of hydrophilic particles/liquids, retarding evaporation of water from the inside and penetration of molecules from the outside. Therefore, the protective function of the skin presents a formidable obstacle and limits the number of drugs that can be delivered transdermally.
Currently, testing one's own glucose concentration is very common and involves a drop of blood taken from the finger tip, which is analyzed via a glucose sensor. This method is not continuous and therefore diabetic people, of type 1 and 2, may experience very often either a state of hypoglycemia or hyperglycemia or both of them many times a day. Moreover, a state of hypoglycemia or hyperglycemia even during a short period of time, but repeated very often, has a disastrous effects on the state of health of the diabetic person. These repeated states might lead in the long term, even if the concentration of glucose is controlled by the abovementioned method, to a deterioration of renal and/or liver functioning and can lead to other deteriorations such as cardiovascular complications. Therefore, new devices which can monitor continuously the concentration of glucose in the body in order to maintain this concentration inside its normal threshold values are much needed.
Presently, there is some breakthrough in this regard in the market. All the technologies available actually are invasive since the sensor is to be placed under the skin and are not very accurate since they need routine calibration and do not last more than a week. This is the case of Navigator® commercialized by Abbott. The Navigator® product is not the only invasive device present in the market, but all of known products lack accuracy, need calibration and do not last for a long period of time. According to the definition of the non-invasive technique, one of the principal requirements is that the sensor should be placed outside the body.
Several patents have proposed that the level of glucose in blood can be monitored by measuring the level of glucose in interstitial fluid. In order to obtain a sample of interstitial fluid, the barrier function of the stratum corneum must be overcome. U.S. Pat. No. 4,775,361, discloses a method of ablating the stratum corneum of a region of the skin of a subject by using pulsed laser light of a wavelength, pulse length, pulse energy, pulse number, and pulse repetition rate sufficient to ablate the stratum corneum without significantly damaging the underlying epidermis. This method should lead to the contact of the sensor with the interstitial fluid. However, its accuracy is very questionable due to the fact that the interstitial fluid is present inside the body under a negative pressure or its movement is restricted, therefore, this kind of method should be comparable to those based on the perforation of the finger in order to obtain a drop of blood.
U.S. Pat. No. 5,423,803, discloses a process for the removal of superficial epidermal skin cells, i.e., stratum corneum, in the human skin. A contaminant having a high absorption in at least one wavelength of light is topically applied to the surface of the skin. Some of the contaminant is forced to infiltrate into spaces between superficial epidermal cells. The treated skin section is illuminated with short laser pulses at the above wave-length, with at least one of the pulses having sufficient energy to cause some of the particles to explode tearing off the superficial epidermal cells. The contaminants include 1 micron graphite particles and the laser used is a Nd:YAG laser. Again, this method should lead to the contact of the sensor with the interstitial fluid. However, its accuracy is very questionable due to the fact that the interstitial fluid is present inside the body under a negative pressure or its movement is restricted, therefore, this kind of method should be comparable to those based on the perforation of the finger in order to obtain a drop of blood.
WO 94/09713 discloses a method for perforating skin comprising the steps of (a) focusing a laser beam in the shape of an ellipse at the surface of the skin with sufficient energy density to create a hole at least as deep as the keratin layer and at most as deep as the capillary layer; and (b) creating at least one hole, each hole having a width between 0.05 and 0.5 mm and a length of equal to or less than 2.5 mm. Again, this method should lead to the contact of the sensor with the interstitial fluid. However, its accuracy is very questionable due to the fact that the interstitial fluid is present inside the body under a negative pressure or its movement is restricted, therefore, this kind of method should be comparable to those based on the perforation of the finger in order to obtain a drop of blood. Another problem associated with this method is the depth of the obtained hole which cannot be controlled therefore leading to the contact of the sensor with the interstitial fluid and the whole blood.
Minimally-invasive methods and apparatus for measuring the glucose level have been produced. For example U.S. Pat. No. 5,730,714 describes a method and a device which uses iontophoresis which employs in this case a constant low voltage and a constant current for as long as the measurement is carried out which may be uncomfortable or at least inconvenient for the subject. EP Patent No. 0889703 describes a method and a device which uses radiation for analyte detection. Both methods described are not accurate since they create an ionic movement in the interstitial fluid near the electrode where the analysis should be carried out. Even if the electrode determines the concentration, it does not represent the same concentration as in the whole interstitial fluid.
Different methods are associated with the transdermal drug delivery. These methods include iontophoresis and electroporation. Thus, one of the possibilities to temporarily breach the barrier function of the skin is by using electroporation, thereby creating aqueous pathways across lipid-based structures of the stratum corneum. Therefore, the electroporation must be repeated on the same part of the skin since the aqueous pathways are reversible and the openings are lost in a very short time. Moreover, the openings are very small in terms of diameter that it is nearly impossible to obtain the interstitial fluid with all of its components and the quantity obtained even with the help of a strong suction is very small if not absent.
Iontophoresis relies on the active transportation of a drug through the skin subjected to an electric field using a simple galvanic current and it is known that iontophoresis typically delivers 100 times less drug quantity than an injection but provides higher local concentrations than oral administration. In iontophoresis, the potential pathways for ingredients to penetrate are restricted, forcing the majority of drugs to permeate the skin via appendageal pores such as hair follicles and sweat glands. These routes only account for about 0.1% of the skin's surface, making drug delivery via iontophoresis inefficient when a large area of tissue requires treatment. Moreover, penetration via the appendages is slow. Moreover, the principle of transdermal drug delivery via ionotophoresis relies also on the diffusion of the species from the more concentrated side to the less concentrated side or above the skin. In the case of the interstitial fluid extraction, there is no diffusion which limits largely the flow of the interstitial fluid, due to its presence in the body under a negative pressure comparatively to the blood which is present under a positive pressure, unless a heavy suction is used and the openings are large and permanent.
In contrast, the number of transdermal pathways, available via electroporation, is over 500 times greater than with iontophoresis. In order to improve the absorption of drugs and to defeat the protective qualities of the stratum corneum, iontophoresis device manufacturers sometimes recommend removing the epidermis via microdermabrasion. While this may seem to enhance the permeability, this step is not required with electroporation. Electroporation, also known as electropermeabilization, is a term used to describe the permeabilization of the cell membrane as a consequence of the application of certain short and intense electric fields across the cell membrane, the cells or the tissues.
Electroporation uses high voltage electric perturbation and result in the re-orientation of the lipid layer to form hydrophilic pores or microconduits. High-voltage pulsing has been shown to enhance transport into or across the skin for compounds ranging in size from small ions such as Na+ and Cl− for example, to moderate sized molecules such as calcein, sulforhodamine, metoprolol, macromolecules such as heparin, oligonucleotides, or latex microspheres of micron dimensions. Again, the principle of transdermal drug delivery via the electroporation relies also on the diffusion of the species from the more concentrated side to the less concentrated side. In the case of the interstitial fluid extraction, there is no diffusion which limits largely the flow of the interstitial fluid unless a heavy suction is used and the openings are large and permanent.
DermaWave No-Needle Mesotherapy System™ uses short, intense electric pulses that alter the electrical potential of the upper layer of the skin and form aqueous pores in the membrane. These pores, or microconduits, are numerous providing the opportunity to deliver compounds evenly into the tissue without the need to alter, change or remove the stratum corneum under only one condition which is the diffusion of the compounds from the more concentrated zone (above the skin) to the less concentrated zone (under the skin). Electroporation proceeds in a domino like manner across the tissue or the upper layer of the skin with the strongest effect being directly beneath the drug application accessory. Some device manufacturers utilize separate accessories for the delivery of electrical pulses and application of topicals. This technique has some problems, since efficient electroporation requires that the electrical energy is delivered in a consistent manner to the tissue with simultaneous delivery of medication. Microconduits return to pre-treatment size after a few milliseconds when the pulses are turned off and the dilation time may be augmented by increasing the duration of the pulsing waveform. However, to achieve maximum transport potential, treatment strategy requires that the applicator is in relatively continuous contact with the tissue area to be treated.
It is disclosed in WO 00/78207 that reverse/reversible electroporation of the skin can be used to have access to a sample of interstitial fluid. Electroporation when carried out using high voltage electric pulse is therefore a very versatile method of reversibly disrupting the stratum corneum. Short pulses (microsecond to millisecond) of pulse field strength sufficient to create a transmembrane potential of more than 0.5 volts cause the capacitive breakdown of the membrane dielectric, leading to transient permeability increases until the membrane recovers sometime after the pulse. Electrically, the skin can be modeled as resistors and capacitors in parallel with most of the resistance residing in the stratum corneum. Thus, if the skin is exposed to an electric pulse, most of the pulse voltage would fall across the stratum corneum, making it the site of the electroporation. Theoretical models of the application of pulsed voltage on the skin suggests that the electropores are created in the lipid lamella and corneocyte membranes such that ions, lipophilic liquid or in general the interstitial fluid with the majority of its components may flow straight through the openings in the stratum corneum if and only if a negative pressure is applied above the treated skin. Therefore, due to the fact that the interstitial fluid has a negative pressure inside the body, it is impossible to drive the interstitial fluid out of the body without the help of a strong suction. Depending on the amplitude of the applied pulse, these electropores might be too small and too uncommon to be detected, being only a few nanometers in diameter and last only a few milliseconds to a few minutes depending on the process of the electroporation.
The openings on the skin obtained by reverse electroporation as described in WO 00/78207 last for a few seconds to a few minutes and the openings have small diameters, in the order of nanometers. Moreover, the reverse or reversible electroporation alone cannot lead to the extraction of interstitial fluid in order to analyze one analyte or a plurality of analytes in the interstitial fluid. Therefore, an enhancement or a combination of enhancements is used to further help the extraction of the interstitial fluid. Such enhancement is described in the art in WO 00/78207 where the electrodes are immersed in different solutions in order to enhance the diffusion of different analytes from the interstitial fluid to the solutions surrounding the electrodes. It is clear that this technique only add more difficulties to analyze one analyte or a plurality of analytes than the difficulties it solves.
Another example of the use of reversible electroporation is the method described in WO 01/62144 where the extraction of the interstitial fluid is carried out by using reverse or reversible electroporation in conjunction with the use of suction. As clearly described in WO 01/62144, the openings do not last for days but for a few minutes at best. Liposomes are thus used to enhance the permeabilization of the skin in WO 01/62144. Moreover, even with the use of liposomes in order to increase the duration of the openings, the openings have small diameters in the order of nanometers and the concentration of the analytes in the extracted interstitial fluid does not reflect those in the interstitial fluid in the body as has been described clearly in WO 01/62144.
It would thus be desirable to be provided with a device that can continuously monitor the concentration of glucose in the body, in a non invasive manner and that can last for a long period of time between calibrations.